Browsing by Subject "adhesion"

Abstract Faculty: Faculty of Agriculture and Forestry* and Faculty of Medicine *coordination Degree programme: Master′s program in Microbiology and Microbial Biotechnology Author: Johanna Potila Title: Characterization of potentially therapeutic bacteria from a healthy fecal donor. Level: Master′s thesis Month and year: August 2023 Number of pages: 40 Keywords: Clostridioides difficile, dysbiosis, FMT, next-generation probiotics, adhesion, anti-inflammatory Supervisors: PhD Kaisa Hiippala, PhD, Docent Reetta Satokari and PhD Pauliina Lankinen Where deposited: E-thesis University of Helsinki Abstract: Recurrent Clostridioides difficile infection (rCDI) is a healthcare-associated infection related to antibiotic use, that causes significant morbidity and mortality. Fecal microbiota transplantation (FMT) is the most effective treatment for rCDI and it is successful in nearly 90% of patients. However, there are some risks related to FMT use such as the potential risk of transferring pathogens or other phenotypes despite donor screening. Defined bacterial mixtures consisting of endogenous commensal gut microbes with beneficial properties could be used instead of FMT to mitigate the risks and improve the availability of the treatment. 12 bacterial strains previously isolated from a healthy fecal donor were characterized in this study. At first, oxygen tolerance and culturability of the isolates in several different media were examined. The second aim was to investigate if these isolates are safe for bacteriotherapeutic use by testing hemolytic properties, antibiotic susceptibilities and proinflammatory properties. The third objective was to investigate potential beneficial properties such as adherence of the isolates to mucus and epithelial cell lines and anti-inflammatory effects on epithelial cells. Caco-2 and HT-29 cell lines were used as a model of intestinal epithelial cells. Growth was abundant on standard brain heart infusion (BHI) medium supplemented with 0,5% yeast extract and more than half of the isolates tolerated the 4-hour oxygen exposure. These results suggest that many of the strains have good production characteristics. All 12 isolates were non-hemolytic and most of them were susceptible to many commonly used anti-microbials such as amoxycillin/clavulanic acid and piperacillin/tazobactam. Low induction of interleukin-8 (IL-8) release from HT-29 cells was observed for all the isolates which indicates no pro-inflammatory effect. These safety tests suggest that the isolates are safe for therapeutical use. Adhesion to mucus and intestinal epithelial cells (HT-29, Caco-2) was low to moderate (2-7%), which can potentially promote their colonization in the gut. No attenuation of Escherichia coli lipopolysaccharide (LPS)-induced IL-8 release from HT-29 cells was observed, which indicates that characterized strains do not have anti-inflammatory effects on epithelial cells. However, it is likely that they have some other important roles in the gut e.g., in cross-feeding networks and can thus help with restoration of a healthy, diverse gut ecosystem. In conclusion, the characterized isolates could be suitable for bacteriotherapeutic use in the treatment of rCDI.

Surface (S-) layers, structural entities that surround the cell envelope of various bacteria, are comprised of a porous lattice of identical protein or glycoprotein subunits. Interestingly, the S-layer is able to promote adherence to host epithelial cells in a variety of Lactobacillus species. L. amylovorus DSM 16698, a strain of porcine origin, encodes at least three putative types of S-layer proteins in its genome sequence. In this study the surface structure of L. amylovorus DSM 16698 strain and the adhesion properties of its S-layer proteins to porcine intestinal epithelial IPEC-1 cell line were examined based on preliminary results. In addition, host receptors potentially specific for S-layer proteins were isolated from IPEC-1 cells. Cloned recombinant S-layer proteins rSlpA and rSlpB of DSM 16698 were reassembled onto fluorescent-labeled L. amylovorus cell wall extracts as a means to mimic the native S-layer lattice structure. Adhesion between the reassembled recombinant S-layer complexes and IPEC-1 cells was assessed qualitatively by microscopy and quantitatively by measuring fluorescence intensity. Results from in vitro adhesion assays indicate that the rSlpA and rSlpB proteins both mediated the adherence of the L. amylovorus DSM 16698 strain to porcine intestinal epithelial cells. Antibodymediated adhesion inhibition experiments were also performed, in which the two rSlps were pretreated with their specific anti-rSlp serum, and showed that adhesion between the rSlps and IPEC-1 cells could be inhibited by the antibody treatment. Moreover, by using fluorescent-labeled rSlp-specific antibody, the surface structure of L. amylovorus cells was microscopically examined. With this immunofluorescent technique, the SlpA and SlpB proteins were both observed to localize on the cell surface and exhibit a similar distribution pattern. Putative S-layer host cell receptors were isolated from the interaction between the reassembled rSlp/cell wall complexes and IPEC-1 derived membrane proteins using a SDS-PAGE-based system. Receptor isolation experiments resulted in repeatedly the same protein profile. It has previously been shown that L. amylovorus DSM 16698 attaches to IPEC-1 cells, but the identities of surface-localized components that mediate this microbe-host interaction had yet to be determined. In this present study, S-layer proteins were found to be an important mediator in the interaction between L. amylovorus DSM 16698 and a porcine epithelial cell line. Additionally, it was shown how S-layer proteins are localized on the surface of L. amylovorus DSM 16698 cells.

Health benefits associated with the ingestion of certain lactobacilli known as probiotics have increased the research and incorporation of these bacteria into food products. Lactobacillus rhamnosus GG is a well-known and studied probiotic organism. Upon ingestion, probiotics survive acid and bile stress and then adhere to the epithelial cell walls to elicit health benefits. Adhesion promotes interaction between probiotic cells and epithelial cells which is necessary for probiotics to confer health benefits. Adhesion is also the first step in biofilm formation which aids adaptability and cell-cell interaction. The aim of this work was to investigate the effect of different carbohydrates on the biofilm formation and antigenicity of Lb. rhamnosus GG. Biofilm formation was performed using 96-well microtiter plating method under elevated carbon dioxide (5% CO2) conditions for 24, 48 and 72 hours in the presence of fourteen different carbohydrates. Certain carbohydrates were found to promote the biofilm formation. The expressed antigenic proteins at the cell surface of biofilms from these carbohydrates were also isolated and investigated using 1DE immunoblot analysis. Four carbohydrates were shown to markedly increase the biofilm of Lb. rhamnosus GG under the indicated conditions. For three of the tested carbohydrates the most efficient biofilm formation was obtained after 48 hours of cultivation, whereas for one of the carbohydrates longer time was required to achieve the same biofilm formation efficiency. One dimensional gel electrophoresis coupled with immunoblotting using antibodies raised against whole Lb. rhamnosus GG cells indicated that the increased biofilm formation is closely associated with the increased surface antigenicity. The obtained findings suggest that certain carbohydrates have a central role in stimulating biofilm mode of growth as well as improving the probiotic features of Lb. rhamnosus GG strain.